Ten small nuclear ribonucleoproteins (snRNPs) in the vertebrate nucleoplasm contribute to the maturation of pre-messenger RNAs as components of the splicing or 3'-end formation machinery. In contrast, approximately two hundred small nucleolar RNPs (snoRNPs) direct cleavages and specific nucleotide modifications, converting the pre- ribosomal RNA (rRNA) into mature 18S, 5.8S and 28S rRNAs. Additional snRNPs (of unknown localization) direct the modification of other snRNAs. The functions, mechanisms of action and biogenesis of these snRNPs and snoRNPs will be investigated using primarily Hela in vitro extracts and the Xenopus oocyte. For the U1, U2, U4/U6 and U5 snRNPs of the major spliceosome, photoactivatable nucleotides will be introduced into the snRNAs to analyze their molecular contacts during splicing. The precise functions of modified nucleotides in these snRNAs will be identified and their biogenesis investigated, particularly by establishing how many are directed by other snRNA guides. For the newly characterized, low-abundance AT-AC spliceosome, both its salient differences compared to the major spliceosome and why it has been maintained in evolution will be probed. AT-AC spliceosome assembly will be dissected to ask why the U11/U12 snRNP acts as a complex and to discover whether AT-AC specific exonic enhancers exist. Analyses of in vivo pre-mRNA populations will establish whether AT-AC intron removal is rate-limiting. Both the necessity for alternative splicing (AT-AC versus major intron removal) of the prospero gene transcript during Drosophila development and its regulation will be investigated. The machinery that fashions the 3' ends of histone mRNAs will be dissected by characterizing the protein(s) that allow measurement along the single-stranded pre-mRNA and by investigating whether the cleavage factor is the same as that required for polyadenylation. SnoRNPs that direct rRNA 2'-O-methylation will be purified to characterize the methylase and an in vitro methylation reaction employing snoRNA-rRNA chimeras will be developed. Nucleotide analog interference mapping (NIAM) will be used to identify the important RNA determinants. Possible coordination between snoRNPs that guide methylation of a particular neighborhood of 28S will be probed. To illuminate why all snoRNA host genes are 5' TOP genes, a nuclear role for 5' TOP sequences that might contribute to the release of intron-encoded snoRNAs will be sought. Perturbations in the biogenesis and functioning of snRNPs and snoRNPs are determinants in certain human diseases and perhaps aging.